Aryl Hydrocarbon Receptor mRNA Expression Research Articles

Exploring the role of air pollution on the development of atherosclerosis disorder in human endothelial cells

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): QU - NSPP Introduction Atherosclerosis, the main cause of cardiovascular and cerebrovascular diseases, is a global health threat. Rising cardiovascular disease prevalence, unexplained by traditional risk factors, prompts exploration of the impact of occupational exposure to environmental toxins as a risk factor. benzo[a]pyrene (BaP) is a polyaromatic compound that mediate its toxicity through activation of the Aryl hydrocarbon receptor (AhR). The molecular mechanisms of involvement of environmental toxins and the role of AhR/CYP1A pathway in atherosclerosis development remain not fully investigated. Purpose The current study explores the AhR pathway activation in endothelial cells upon exposure to environmental toxins in order to elucidate the molecular mechanisms of atherosclerotic cardiovascular diseases and plaque formation through investigating pro-inflammatory (CYP1A1, CYP1B1, TNF-a) and pro-apoptotic markers (BCL-2, Caspase-3). Methods EA. hy926 cell line underwent treatment with increasing concentrations of BaP (0-20 microM), as well as 10nm of TCDD, to confirm the results. DSMO was used as a control. Total RNA and protein were isolated to investigate mRNA expression and protein expression of AhR, CYP1A1, CYP1B1, as well as caspase-3, Bcl-2, and TNF-α using RT-PCR and Western Blot analysis. Statistical analysis was done using one-way ANOVA. Results The results showed that BaP induce significant dose-dependent upregulation of AhR and target genes (CYP1A1, CYP1B1). At 20uM, expression of AhR (3-fold), CYP1A1 (2.5-fold) and CYP1B1 (2-fold) was significantly high as compared to their respective controls and low doses. As for apoptotic markers, Bcl-2 was significantly downregulated at the highest concentrations 10uM and 20uM (0.7- and 0.6-fold, respectively). Caspase-3 showed no significant change but exhibited upregulation across all doses. TCDD 10nM significantly upregulated AhR (2.0-fold), CYP1A1 (1.5-fold), CYP1B1 (1.7-fold). Bcl-2 downregulated (0.6-fold), TNF-α significantly upregulated (1.8-fold) at 10nM. Caspase-3 increased 2-folds but was not statistically significant. Conclusion This study illustrated that AhR inducers, such as BaP, can accelerate the pathological progress of atherosclerosis by evoking oxidative stress and inflammation response molecules in human endothelial cells, demonstrated by the reduced expression of anti-apoptotic markers and the increased expression of inflammatory markers.

P05 The aryl hydrocarbon receptor pathway is dysfunctional in atopic dermatitis

Abstract Introduction and aims Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by epidermal barrier disruption and type-2 immune dysregulation. While treatment has improved with targeted biologics, clinical response varies, highlighting the need for additional therapeutic strategies. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor and environmental sensor, which maintains skin barrier integrity and homeostasis by reducing inflammation. Upon activation, AHR induces the expression of CYP1A1, a biomarker of pathway activation and critical for AHR ligand metabolism. Tapinarof, a topical AHR agonist, is approved for psoriasis and is effective in phase III clinical trials in AD, highlighting the importance of this pathway in inflammatory skin disease. Nevertheless, AHR expression is increased in AD skin, leading to the hypothesis that the pathway is dysregulated, thus not exerting its anti-inflammatory effect. Methods Here we begin to perform a global investigation of the AHR pathway in AD. We measured AHR and CYP1A1 mRNA expression by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in healthy and AD skin, with spatial mRNA expression assessed by RNAscope. We also investigated AHR mRNA expression in interleukin (IL)-4 stimulated (100 ng mL−1) normal human epidermal keratinocytes (NHEKs) using qRT-PCR. Results We found that AHR mRNA is significantly upregulated in lesional AD (n = 6) compared with healthy skin (n = 9; P < 0.05), whereas CYP1A1 mRNA expression shows a downward trend in lesional AD compared with healthy skin (P = 0.063). The AHR expression pattern differs in lesional AD (n = 7) compared with healthy skin (n = 8), with AHR localizing in the epidermal basal layer. Finally, IL-4 significantly upregulated AHR mRNA (n = 4 wells; P < 0.001) expression in NHEKs compared with unstimulated cells. Conclusions Overall, our data have uncovered a dysfunctional AHR pathway in AD, with increased AHR expression, possibly driven by the AD inflammatory milieu, but reduced pathway activation. Further investigations are required to fully dissect this pathway and maximize its potential as a therapeutic target for AD.

Oxidative stress and inflammation are mediated via aryl hydrocarbon receptor signalling in idiopathic membranous nephropathy.

Membranous nephropathy (MN) patients are diagnosed by the presence of phospholipase A2 receptor (PLA2R) before they progress to renal failure. However, the subepithelium-like immunocomplex deposit-mediated downstream molecular pathways are poorly understood. The aryl hydrocarbon receptor (AHR), NF-ƙB and Nrf2 pathways play central roles in the pathogenesis and progression of chronic kidney disease. However, their mutual effects on MN require further examination. Thus, we investigated the effect of AHR signalling on the NF-ƙB and Nrf2 pathways in IMN patients, cationic bovine serum albumin (CBSA)-injected rats and zymosan activation serum (ZAS)-treated podocytes. IMN patients show significantly decreased serum total protein and albumin levels, increased urine protein levels and intrarenal IgG4 and PLA2R protein expression in glomeruli compared with controls. IMN patients exhibited increased mRNA expression of intrarenal AHR and its target genes, including CYP1A1, CYP1A2, CYP1B1 and COX-2. This increase was accompanied by significantly upregulated protein expression of CD3, NF-ƙB p65 and COX-2 and significantly downregulated Nrf2 and HO-1 expression. Similarly, CBSA-induced rats showed severe proteinuria and activated intrarenal AHR signalling. This was accompanied by significantly upregulated protein expression of intrarenal p-IκBα, NF-κB p65 and its gene products, including COX-2, MCP-1, iNOS, 12-LOX, p47phox and p67phox, and significantly downregulated protein expression of Nrf2 and its gene products, including HO-1, catalase, GCLC, GCLM, MnSOD and NQO1. These results were further verified in ZAS-induced podocytes. Treatment with the AHR antagonist CH223191 and AHRsiRNA significantly preserved podocyte-specific protein expression and improved the NF-ƙB and Nrf2 pathways in ZAS-induced podocytes. In contrast, similar results were obtained in ZAS-induced podocytes treated with the NF-ƙB inhibitor BAY 11-7082 and NF-κBp65 siRNA. However, neither method had a significant effect on AHR signalling. Collectively, these results indicate that the NF-ƙB pathway is a downstream target of AHR signalling. Our findings suggest that blocking AHR signalling inhibits oxidative stress and inflammation, thereby improving proteinuria and renal injury.

Lactobacillus species ameliorate membranous nephropathy through inhibiting the aryl hydrocarbon receptor pathway via tryptophan-produced indole metabolites.

Membranous nephropathy (MN) is an immune-mediated glomerular disease in adults. Antibody- and antigen-bonding mechanisms have been largely clarified, but the subepithelium immune complex deposition-mediated downstream molecular mechanisms are currently unresolved. Increasing evidence has suggested that gut microbiota contribute to MN pathogenesis. In this study, we identified alterations in faecal gut microbiota and serum metabolites that mediate an aryl hydrocarbon receptor (AhR) mechanism in cationic bovine serum albumin (CBSA)-induced MN rats and in patients with idiopathic MN (IMN). Impaired renal function correlated with the relative abundance of reduced faecal probiotics, Lactobacillus and Bifidobacterium, and altered serum levels of tryptophan-produced indole derivatives (TPIDs) in MN rats. Further results showed that reduced relative abundance of five probiotics, namely Lactobacillus johnsonii, Lactobacillus murinus, Lactobacillus vaginalis, Lactobacillus reuteri and Bifidobacterium animalis, positively correlated with decreased levels of indole-3-pyruvic acid, indole-3-aldehyde and tryptamine and negatively correlated with increased levels of indole-3-lactic acid and indole-3-acetic acid in serum of MN rats. Altered five probiotics and five TPIDs also were observed in patients with IMN. Further studies showed that MN rats exhibited a significant increase in intrarenal mRNA expression of AhR and its target genes CYP1A1, CYP1A2 and CYP1B1, which were accompanied by protein expression of down-regulated cytoplasmic AhR, but up-regulated nuclear AhR, in MN rats and IMN patients. Activation of the intrarenal AhR signalling pathway may involve five TPIDs. These data suggest that gut microbiota could influence MN through TPIDs that engage host receptors.

Effects of enhancing the expression of aryl hydrocarbon receptor in post-traumatic mice macrophages on the inflammatory cytokine level and bactericidal ability

Objective: To explore the expression pattern of aryl hydrocarbon receptor (AhR) in mice peritoneal macrophages (PMs) after major trauma and analyze the effects of enhanced AhR expression on the inflammatory cytokine level and bactericidal ability after trauma. Methods: The experimental study method was used. Forty 6-8-week-old male C57BL/6J mice (the same mouse age, sex, and strain below) were divided into control group, post trauma hour (PTH) 2 group, PTH 6 group, and PTH 12 group according to the random number table (the same grouping method below), with 10 mice in each group. Mice in the latter 3 groups were constructed as severe trauma model with fracture+blood loss, while mice in control group were left untreated. The primary PMs (the same cells below) were extracted from the mice in control group, PTH 2 group, PTH 6 group, and PTH 12 group when uninjured or at PTH 2, 6, and 12, respectively. Then the protein and mRNA expressions of AhR were detected by Western blotting and real-time fluorescence quantitative reverse transcription polymerase chain reaction, respectively, and the gene expressions of AhR signaling pathway related molecules were analyzed by transcriptome sequencing. Twenty mice were divided into control group and PTH 6 group, with 10 mice in each group, and the PMs were extracted. The level of ubiquitin of AhR was detected by immunoprecipitation. Twelve mice were divided into dimethyl sulfoxide (DMSO) alone group, PTH 6+DMSO group, MG-132 alone group, and PTH 6+MG-132 group, with 3 mice in each group. After the corresponding treatment, PMs were extracted, and the protein expression of AhR was detected by Western blotting. Twenty mice were constructed as PTH 6 model. Then, the PMs were extracted and divided into empty negative control adenovirus (Ad-NC) group and AhR overexpression adenovirus (Ad-AhR) group. The protein expression of AhR was detected by Western blotting at 36 h after some PMs were transfected with the corresponding adenovirus. The rest cells in Ad-NC group were divided into Ad-NC alone group and Ad-NC+endotoxin/lipopolysaccharide (LPS) group, and the rest cells in Ad-AhR group were divided into Ad-AhR alone group and Ad-AhR+LPS group. The expressions of interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) in the cell supernatant were detected by enzyme-linked immunosorbent assay at 12 h after the corresponding treatment (n=6). Twenty mice were obtained to extract PMs. The cells were divided into control+Ad-NC group, PTH 6+Ad-NC group, control+Ad-AhR group, and PTH 6+Ad-AhR group, and the intracellular bacterial load was detected by plate spread method after the corresponding treatment (n=6). Data were statistically analyzed with one-way analysis of variance, least significant difference test, analysis of variance for factorial design, and independent sample t test. Results: Compared with 1.16±0.28 of control group, the protein expressions of AhR in PMs in PTH 2 group (0.59±0.14), PTH 6 group (0.72±0.16), and PTH 12 group (0.71±0.17) were all significantly decreased (P<0.05). The overall comparison of the difference of AhR mRNA expression in PMs among control group, PTH 2 group, PTH 6 group, and PTH 12 group showed no statistical significance (P>0.05). The AhR signaling pathway related molecules included AhR, AhR inhibitor, cytochrome P450 family member 1b1, cytochrome P450 family member 11a1, heat shock protein 90, aryl hydrocarbon receptor-interaction protein, and heat shock protein 70 interaction protein. The heat shock protein 90 expression of PMs in PTH 2 group was higher than that in control group, while the expressions of other molecules did not change significantly after trauma. Compared with that in control group, the level of ubiquitin of AhR in PMs in PTH 6 group was increased. Compared with that in DMSO alone group, the protein expression of AhR in PMs in PTH 6+DMSO group was decreased, while that in PMs in MG-132 alone group had no significant change. Compared with that in PTH 6+DMSO group, the protein expression of AhR in PMs in PTH 6+MG-132 group was up-regulated. At transfection hour 36, compared with that in Ad-NC group, the protein expression of AhR in PMs in Ad-AhR group was increased. At treatment hour 12, compared with those in Ad-NC+LPS group, the expressions of IL-6 and TNF-α in PM supernatant of Ad-AhR+LPS group were significantly decreased (with t values of 4.80 and 3.82, respectively, P<0.05). The number of intracellular bacteria of 1×106 PMs in control+Ad-NC group, PTH 6+Ad-NC group, control+Ad-AhR group, and PTH 6+Ad-AhR group was (3.0±1.8), (41.8±10.2), (1.8±1.2), and (24.2±6.3) colony forming unit, respectively. Compared with that in PTH 6+Ad-NC group, the number of intracellular bacteria of PMs in PTH 6+Ad-AhR group was significantly decreased (t=3.61, P<0.05). Conclusions: Ubiquitin degradation of AhR in PMs of mice after major trauma results in decreased protein expression of AhR. Increasing the expression of AhR in post-traumatic macrophages can reduce the expressions of LPS-induced inflammatory cytokines IL-6 and TNF-α, and improve the bactericidal ability of macrophages after trauma.

Indol-3-Carbinol ameliorates colitis in mice by AhR-mediated production of antimicrobial peptides and mucins

Abstract Indole-3-carbinol (I3C) is a dietary compound that acts as a ligand for the aryl hydrocarbon receptor (AhR), an important sensor for environmental polyaromatic chemicals and a regulator of the immune response. The pathogenesis of colitis involves multiple interactions between microbial dysbiosis, dysregulation of the host immune response, environmental factors, and host genetic profile. Indeed, studies have shown defective expression of AhR and antimicrobial peptides (AMPs) particularly defensins in inflammatory bowel disease (IBD) patients. Here, we investigated how I3C influences the production of AMPs, particularly b-defensins, through AhR and colonic microbiota composition in Anti-CD40 or dextran sulfate sodium (DSS)-induced colitis. Our analysis found that I3C attenuates colitis through AhR activation leading to increased expression of murine b-defensins (mBDs) by Colonic Epithelial Cells, resulting in the restoration of healthy gut microbiota and prevention of colonic inflammation. We analyzed the colonic expression of AMPs and mucins. Our analysis found that I3C significantly increased the mRNA expression of AhR, AMPs such as mBDs, Reg4, and mucins, when compared to vehicle-treated mice with colitis. Similarly, the mRNA levels of AhR, AMPs, and mucins were significantly increased in DSS+I3C-treated colon adenocarcinoma cells MC-38 (murine) and Caco2 (human) when compared to DSS-treated cells. To conclude, our findings demonstrate that I3C inhibits colitis primarily through AhR-mediated induction of AMPs and protective mucins, resulting in the enrichment of healthy gut microbiota (This work was supported in part by NIH grants R01ES030144, P01AT003961, P20GM103641, and R01AI123947, R01AI160896). This work was supported in part by NIH grants R01ES030144, P01AT003961, P20GM103641, and R01AI123947, R01AI160896

Crosstalk between the aryl hydrocarbon receptor (AhR) and the peroxisome proliferator-activated receptor gamma (PPARγ) as a key factor in the metabolism of silver nanoparticles in neuroblastoma (SH-SY5Y) cells in vitro

The potential usefulness of silver nanoparticles (AgNPs) in anticancer therapy has been postulated for many years. However, little is known to date about the exact impact of such NPs on intracellular detoxication pathways. Therefore, the aim of this study was to determine the impact of AgNPs on the AhR-PPARγ-CYP1A1 pathway in neuroblastoma (SH-SY5Y) cells. The obtained results showed a decrease in the metabolic activity of the SH-SY5Y cells at the 50 and 100 μg/mL concentrations with an increase in caspase-3 activity. An increase in the intercellular ROS production was observed at the 1 and 10 μg/mL concentrations. The co-treatment of the AgNP-treated cells with the AhR and PPARγ inhibitors abolished the effect of the tested AgNPs in the SH-SY5Y cells. In turn, the CYP1A1 activity assay showed a decrease in this parameter in the AgNP-treated cells. Moreover, the gene expression analysis demonstrated that AgNPs were able to increase the AhR and CYP1A1 mRNA expression and decrease the PPARγ gene expression after the 6-h treatment. In turn, an increase in the AhR and PPARγ protein expression was observed after 24 h. Summarizing, the study shows for the first time that AgNPs with a 5-nm diameter size are able to exert a cytotoxic effect on SH-SH5Y cells in a ROS-dependent manner affect the AhR-PPARγ-CYP1A1 pathway inter alia by inhibiting the activity of CYP1A1. This is important due to given present research approaches using such NPs as enhancer agents in the modern PPARγ inhibitor-based anticancer therapy.

337 Multi-level dysregulation of the AHR/CYP1A1 axis in psoriasis

The aryl hydrocarbon receptor (AHR) is an environmentally-regulated transcription factor with homeostatic and anti-inflammatory roles in skin. AHR activation leads to expression of the cytochrome P4501 (CYP1) enzyme family, which provides critical negative feedback by degrading AHR ligands. We have previously shown that excessive CYP1A1 activity impairs AHR anti-inflammatory activity in psoriasis-like skin inflammation in mice, while suppression of aberrant enzymatic activity restores AHR beneficial effect. Moreover, CYP1A1 activity is significantly enhanced in blood cells of psoriasis patients, suggesting that dysregulation of CYP1A1 activity may play a role in psoriasis. Here, we performed a multi-modal investigation of the AHR/CYP1A1 axis in the blood and skin of psoriasis patients and healthy controls, assessing availability of AHR ligands and precursors by mass spectrometry, AHR and CYP1A1 mRNA by qPCR, and CYP1A1 enzymatic activity by EROD assay. Levels of the ligand precursor tryptophan were significantly (p≤0.001) decreased in the serum of psoriasis patients (n=20) versus healthy (n=20). Expression of AHR mRNA was significantly (p≤0.001) decreased in both psoriasis blood and psoriasis lesional (PsL) skin, while CYP1A1 mRNA did not differ in blood but was significantly (p≤0.05) decreased in PsL skin. In vitro culture of skin epidermal sheets with an AHR agonist significantly (p≤0.05) upregulated CYP1A1 mRNA in healthy and psoriasis skin, but, surprisingly, CYP1A enzymatic activity was not increased by AHR ligation in PsL. We are currently studying AHR and CYP1A1 protein expression in the skin at single-cell level by imaging mass cytometry to gain further insights into the complex regulation of the pathway. Taken together, our data show that there is a multi-level dysregulation of the AHR/CYP1A1 axis in psoriasis and suggest a failure of multiple pathway checkpoints that could be targeted to restore AHR beneficial effects in skin inflammation.

Intrarenal 1-methoxypyrene, an aryl hydrocarbon receptor agonist, mediates progressive tubulointerstitial fibrosis in mice.

Recent studies have shown that endogenous metabolites act via aryl hydrocarbon receptor (AhR) signalling pathway in tubulointerstitial fibrosis (TIF) pathogenesis. However, the mechanisms underlying endogenous metabolite-mediated AhR activation are poorly characterised. In this study, we conducted untargeted metabolomics analysis to identify the significantly altered intrarenal metabolites in a mouse model of unilateral ureteral obstruction (UUO). We found that the levels of the metabolite 1-methoxypyrene (MP) and the mRNA expression of AhR and its target genes CYP1A1, CYP1A2, CYP1B1 and COX-2 were progressively increased in the obstructed kidney at Weeks 1, 2 and 3. Furthermore, these changes were positively correlated with progressive TIF in UUO mice. In NRK-52E, RAW 264.7 and NRK-49F cells, MP dose-dependently upregulated the mRNA expression of AhR and its four target genes and the protein expression of nuclear AhR, accompanied by the upregulated protein expression of collagen I, α-SMA and fibronectin, as well as downregulated E-cadherin expression. Consistently, oral administration of MP in mice progressively enhanced AhR activity and upregulated profibrotic protein expression in the kidneys; these effects were partially inhibited by AhR knockdown in MP-treated mice and cell lines. In addition, we screened and identified erythro-guaiacylglycerol-β-ferulic acid ether (GFA), which was isolated from Semen plantaginis, as a new AhR antagonist. GFA significantly attenuated TIF in MP-treated NRK-52E cells and mice by partially antagonising AhR activity. Our results suggest that MP activates AhR signalling, thus mediating TIF through epithelial-mesenchymal transition and macrophage-myofibroblast transition. MP is a crucial metabolite that contributes to TIF via AhR signalling pathway.

AHR gene expression and the polymorphism rs2066853 are associated with clinicopathological parameters in colorectal carcinoma

The relationship between red and processed meat and its risk towardcolorectal carcinoma (CRC) is not fully explored in literature. Polycyclic aromatic hydrocarbons (PAHs) are procarcinogenic molecules that are ingested with meat cooked at high temperatures. The metabolic conversion of PAHs to carcinogenic diol epoxides is in part mediated by the aryl hydrocarbon receptor (AhR)-dependent induction of CYP1A1. This study aims to examine the expression profiles and polymorphisms of the AHR (aryl hydrocarbon receptor) gene which is involved in the metabolic conversion of PAHs in patients with CRC. Genetic analysis was done in matched cancer and non-neoplastic tissues from 79 patients diagnosed with CRCs. Low AHR mRNA expression was associated with mucinous colorectal adenocarcinoma. Exon 10 of AHR showed that 27% of patients had the rs2066853 single-nucleotide polymorphism resulting in an arginine-to-lysine change at codon 554. This variant was significantly associated with a lower likelihood of perineural invasion, presence of synchronous cancer, and multiple colorectal polyps. Furthermore, rs2066853 individuals were significantly more likely to be of more advanced age and have a more favorable tumor grade and pathological stage. These results imply the pathogenic roles of AHR in PAH-associated colorectal carcinogenesis.

Effects of intranasal instillation of nanoparticulate matter in the olfactory bulb

Nanoparticulate matter activates the aryl hydrocarbon receptor (AhR) pathway in the respiratory system in a process involving the AhR nuclear translocator (ARNT) and cytochrome P450 family 1, member A1 (CYP1A1). We examined changes in AhR-related pathways following intranasal instillation of nanoparticulate matter in the olfactory bulb and cerebral cortex. Twice a day for 5 days per week for 1 week or 2 weeks, 8-week-old Sprague–Dawley rats were intranasally instilled with 10 µL nanoparticulate matter (nano group; n = 36). An equal volume of saline was intranasally instilled in control rats (n = 36). One week after intranasal instillation, olfactory function and Y-maze tests were performed. The expression levels of AhR in the olfactory bulb and temporal cortex were analyzed using western blotting and immunofluorescence assays. The expression levels of AhR, CYP1A1, inducible nitric oxide synthase (iNOS), and five genes encoding cation transporters (ARNT, ATP7B, ATPB1, OCT1, and OCT2) in the olfactory bulb were analyzed using quantitative reverse transcription. The olfactory discrimination capability was reduced in the nano group compared with the control group. Proportional changes in the Y-maze test were not significantly different between the nano and control groups. AhR mRNA and protein expression in the olfactory bulb increased 1.71-fold (P < 0.001) and 1.60-fold (P = 0.008), respectively. However, no significant changes were observed in the temporal cortex. In the olfactory bulb, the expression of ARNT, ATP7B, ATPB1, and OCT2 was downregulated. CYP1A1 and iNOS expression in the olfactory bulb was upregulated compared with that in the temporal cortex. The intranasal instillation of nanoparticulate matter decreased the olfactory discrimination ability, which was accompanied by upregulation of AhR expression and downregulation of cation transporters in the olfactory bulb.

Tris (2,3-Dibromopropyl) Isocyanurate (TDBP-TAZTO or TBC) Shows Different Toxicity Depending on the Degree of Differentiation of the Human Neuroblastoma (SH-SY5Y) Cell Line

Tris (2,3-dibromopropyl) isocyanurate (TDBP-TAZTO or TBC) is a heterocyclic hexabromated flame retardant. It is widely used during the production of many synthetic compounds. High concentrations of TDBP-TAZTO were found in river water, surface sediments, soil, earthworms, and carp tissues. Moreover, it has been shown that this compound can cross the blood–brain barrier and accumulate in the gut and brain of carp. The aryl hydrocarbon receptor (AhR) has been characterized as a multifunctional intracellular sensor and receptor. AhR is an activator of cytochrome P450 1A1 and 1A2, which metabolize various toxic compounds. The aim of the study was to explain how/whether TDBP-TAZTO increases the expression and/or activity of the CYP1A1 enzyme and the AhR and TUBB3 expression during SH-SY5Y cell differentiation. SH-SY5Y cells were differentiated for 7 and 14 days using retinoic acid. Cell viability, ethoxyresorufin-O-deethylase (EROD) activity, and mRNA expression of CYP1A1, AhR, and TUBB3 were assessed. Our experiment showed that, during the differentiation process, the ability of TDBP-TAZTO to induce EROD activity in SH-SY5Y cells subsequently decreased, which may have been an effect of cell differentiation into neurons. Moreover, the results suggest that TDBP-TAZTO can affect the differentiation process. Since no CYP2B6 mRNA expression was detected, the CAR receptor may not be involved in the TDBP-TAZTO mechanism of action. However, more research is needed in this field to elucidate this mechanism precisely.

Bisphenol analogs AF, S and F: Effects on functional characteristics of porcine granulosa cells

In order to replace industrial functions of the restricted endocrine disruptor bisphenol A (BPA), its structural analogs are increasingly employed without adequate assessment of their biological actions. Our study examined effects of the bisphenols AF (BPAF), S (BPS) and F (BPF), on functions of porcine ovarian granulosa cells (GCs) with the focus on viability, steroid production (10-9–10-4M), and expression of factors (10-9–10-5M) important for the follicle development: vascular endothelial growth factor A (VEGFA), matrix metalloproteinase 9 (MMP9), forkhead box O1 (FOXO1), and aryl hydrocarbon receptor (AHR). Cell viability was not impaired by the bisphenol analogs, except for the highest BPAF concentration (10-4M). While the lower concentrations of the bisphenols were without effect, each of them reduced follicle-stimulating hormone (FSH)-induced progesterone synthesis at the highest dose. Estradiol synthesis was sensitive to BPS, inhibitory effects of which were manifested from the concentration of 10-6M. Treatment of GCs with the selected bisphenol concentrations did not result in marked alterations in steroidogenic enzyme expression. Bisphenols did not significantly modulate VEGFA mRNA expression or output either under basal or FSH-stimulated conditions. BPF at 10-5M increased MMP9 expression in FSH-stimulated cells. FSH upregulated FOXO1 expression, however, none of the bisphenols significantly affected FOXO1 levels either in basal or in FSH-stimulated conditions. AHR mRNA expression remained unchanged after bisphenol treatment. Although the significant effects of BPAF, BPS and BPF appeared only at supraphysiological doses, the results obtained indicate that BPA analogs are not inert with regard to ovarian physiology.

Aryl Hydrocarbon Receptor and Autophagy-Related Protein Microtubule-Associated Protein Light Chain 3 Expression in Psoriasis.

BackgroundThe aryl hydrocarbon receptor (AHR) and autophagy are both important to maintain skin homeostasis. However, they are also involved in skin disorders. So far, their roles in psoriasis pathogenesis are unknown.ObjectiveWe studied the immunohistochemical and gene expression of AHR, CYP1A1, and microtubule-associated protein light chain 3 (LC3) in lesional skin of psoriasis patients to determine correlations among them.MethodsWe included 24 psoriasis patients and ten healthy volunteers. Skin biopsies were collected. AHR, CYP1A1, and LC3 protein expression was examined by immunohistochemistry, immunofluorescence, and western blotting. AHR, CYP1A1, LC3, ATG5, BECN1 and Nrf2 mRNA levels were measured by quantitative polymerase chain reaction.ResultsAHR and CYP1A1 protein expression were higher in psoriasis lesional skin than in normal skin. LC3 protein expression was lower in psoriasis lesions than in normal controls. AHR and CYP1A1 protein expression in psoriasis lesions showed significant positive correlations with mean epidermal thickness and inflammatory cell density. Significant negative correlations were noted between LC3 protein expression in psoriasis lesions and the mean epidermal thickness or inflammatory cell density. A significant negative correlation was found between AHR and LC3 expression in psoriatic skin. AHR, CYP1A1 and Nrf2 mRNA expression were upregulated while LC3, ATG5, and BECN1 mRNA were down-regulated, in psoriatic lesional skin compared with normal controls.ConclusionAHR and autophagy could play a role in psoriasis pathogenesis by modifying epidermal hyperproliferation and inflammation. AHR and autophagy regulation are potential therapeutic targets in chronic inflammatory skin diseases.

Increased AHR Transcripts Correlate With Pro-inflammatory T-Helper Lymphocytes Polarization in Both Metabolically Healthy Obesity and Type 2 Diabetic Patients

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor whose transcription activity is regulated by small compounds provided by diet, xenobiotics, and metabolism. It has been proven to be involved in energy homeostasis and inflammation in most recent years. Epidemiologically, exposure to xenobiotic AHR ligands contributes to obesity and type 2 diabetes (T2D). AHR is also the critical transcription factor determining the lineage commitment of pro-inflammatory Th17 and Th22 cells from naïve CD4+ T lymphocytes. It has been well-illustrated in animal models that IL-22, the major effector cytokine of Th17 and Th22 cells, played a major role in the interaction of metabolism and gut microbiota. But there were still missing links between gut microbiota, IL-22, and metabolism in humans. Our previous findings indicated that elevated circulating levels of IL-22 and frequencies of Th22 cells were associated with insulin resistance in both patients with obesity and T2D. Additionally, the hyperactive Th17 and Th22 cells phenotype also correlate with islets β-cell dysfunction in T2D. In this study, we made efforts to determine AHR expressions in peripheral blood mononuclear cells (PBMCs) from patients with T2D and metabolically healthy obesity (MHO). Correlation analyses were conducted to assess the possible link between AHR and the metabolic and inflammatory context. We revealed that mRNA expression of AHR was up-regulated and correlated with the percentage of Th17, Th22 as well as Th1 cells. Elevated plasma levels of IL-22 and IL-17 also correlated with increased AHR transcripts in PBMCs from both MHO and T2D patients. The transcription factor AHR may thus have a plausible role in the interaction between metabolism and pro-inflammatory status of patients in the development of obesity and T2D.

芳香烃受体在特应性皮炎患者外周血及皮损组织中的表达及意义

Objective To detect levels of aryl hydrocarbon receptor (AhR) and its downstream molecules in peripheral blood mononuclear cells (PBMCs) and sera from patients with atopic dermatitis (AD) , and to analyze the correlation of their expression with serum cytokines and the severity of AD. Methods Real-time quantitative PCR (RT-PCR) was performed to analyze mRNA expression of AhR, cytochrome P4501A (CYP1A1) , AhR repressor (AHRR) , AhR nuclear translocator (ARNT) in PBMCs from 29 AD patients and 17 healthy controls, enzyme -linked immunosorbent assay (ELISA) to detect serum levels of interleukin (IL) -1β, IL-6, tumor necrosis factor (TNF) -α, IL-4, IL-22 and AhR in the AD patients, and immunohistochemical study to determine AhR expression in skin lesions of the AD patients and normal skin tissues of 21 patients with pigmented nevus. Measurement data were compared by using unpaired Student′s t test, enumeration data were compared by using chi-square test, and correlations between indices were analyzed by using Pearson correlation analysis. Results The serum level of AhR was significantly higher in the AD group (41.26 ± 4.52 pmol/L) than in the healthy control group (33.73 ± 2.49 pmol/L, t = 6.507, P<0.001) . Compared with the healthy control group, the AD group showed significantly increased mRNA expression of AhR (1.572 ± 0.392 vs. 1.000 ± 0.173, t = 6.819, P = 0.007) , AHRR (2.402 ± 1.716 vs. 1.000 ± 0.788, t = 3.722, P = 0.039) , CYP1A1 (2.258 ± 1.598 vs. 1.000 ± 0.796, t = 3.400, P = 0.002) and ARNT (1.383 ± 0.842 vs. 1.000 ± 0.586, t = 1.653, P = 0.105) in PBMCs. The AhR expression in skin lesions in the AD group was significantly higher than that in normal skin tissues in the control group (0.191 ± 0.041 vs. 0.087 ± 0.017, t = 10.036, P<0.001) . In the AD group, the mRNA expression of AhR in PBMCs was positively correlated with eczema area and severity index score (r = 0.448, P = 0.019) and the serum IL-6 level (r = 0.377, P = 0.046) , and the AHRR mRNA expression was positively correlated with the serum IL-1β level (r = 0.467, P = 0.021) . Conclusion AhR and its downstream molecules were highly expressed in the AD patients compared with healthy controls, and the AhR expression was positively correlated with the serum IL-6 level and AD severity in AD patients, suggesting that the AhR signaling pathway may play a certain role in pathogenesis of AD and AhR may serve as an efficient index for evaluating AD severity. Key words: Dermatitis, atopic; Hydrocarbons, aromatic; Cytochrome P-450 enzyme system; Interleukin-6; Aryl hydrocarbon receptor

The Aryl Hydrocarbon Receptor Is Expressed in Thyroid Carcinoma and Appears to Mediate Epithelial-Mesenchymal-Transition.

Aryl hydrocarbon receptor (AhR) is expected to promote initiation, progression and invasion of cancer cells regulating proliferation, differentiation, gene expression, inflammation, cell motility and migration. Furthermore, an immunosuppressant function of AhR has been recognized. This study evaluated AhR expression and its role in thyroid cancer progression. AhR expression was assessed by qPCR in 107 thyroid cancer samples (90 PTCs, 11 MTCs, 6 ATCs), and by immunohistochemistry in 41 PTCs. To estimate receptor activation, the expression of target genes CYP1A1 and CYP1B1 was measured. AhR functional effects were evaluated in kynurenine-stimulated FTC-133 and BcPap cell lines by analyzing the expression of genes involved in EMT and cell motility. AhR mRNA expression resulted significantly higher in all the analyzed thyroid cancer samples compared to normal thyroid and a statistically significant correlation with CYP1B1 was detected. Kynurenine-stimulated FTC-133 and BcPap showed the activation of a specific AhR-driven EMT program characterized by E-cadherin decrease and SLUG, N-cadherin and fibronectin increase, resulting in boost of cell motility and invasion. This study confirmed the importance of the IDO1-Kyn-AhR pathway in thyroid cancer tumorigenesis, suggesting an AhR pivotal role in mediating an immunosuppressive microenvironment and favoring the acquisition of a mesenchymal phenotype that could promote invasiveness and metastasis.

Resistant starch supplementation effects on plasma indole 3-acetic acid and aryl hydrocarbon receptor mRNA expression in hemodialysis patients: Randomized, double blind and controlled clinical trial.

ABSTRACTIntroduction:Gut microbiota imbalance is linked to high uremic toxins production such as indole-3-acetic acid (IAA) in chronic kidney disease patients. This toxin can activate the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor involved with inflammation. Strategies to restore gut microbiota balance can be associated with reduced production of IAA and its deleterious effects. This study aimed to evaluate prebiotic resistant starch (RS) supplementation effects on IAA plasma levels and AhR mRNA expression in CKD patients on hemodialysis (HD).Methods:This randomized, double-blind and placebo-controlled clinical trial evaluated forty-two stable HD patients allocated in RS (n=22) or placebo (n=20) groups. Patients received, alternately, cookies and sachets containing 16 g/day of RS (Hi-Maize 260®) or manioc flour for four weeks. Fasting pre-dialysis blood samples were collected and IAA plasma levels measured by high performance liquid chromatography. Peripheral blood mononuclear cells were isolated and processed for AhR and nuclear factor kappa B (NF-κB) mRNA expression analyzes by quantitative real-time PCR. Anthropometric and biochemical parameters, as well as food intake were also evaluated.Results:Thirty-one patients completed the study, 15 in the RS group and 16 in the placebo group. Although there was no significant alteration in IAA plasma levels, neither in AhR mRNA expression and NF-κB mRNA expression after RS supplementation, a positive correlation (r=0.48; p=0.03) was observed between IAA plasma levels and AhR expression at baseline.Conclusion:Even though prebiotic RS supplementation did not influence IAA levels or AhR expression, their positive association reinforces a possible interaction between them.

Ethanol Augments Monosodium Urate-Induced NLRP3 Inflammasome Activation via Regulation of AhR and TXNIP in Human Macrophages.

PurposeEthanol elicits several inflammatory responses and affects the innate immune response. The aim of this study was to identify the mechanism by which ethanol affects uric acid-induced NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation by regulation of aryl hydrocarbon receptor (AhR) and thioredoxin-interacting protein (TXNIP).Materials and MethodsHuman myeloid leukemia cells (U937 cells) were used to assess the role of ethanol in NLRP3 inflammasome activation induced by monosodium urate (MSU) crystals. Expression of target molecules, such as NLRP3 inflammasome components, AhR, and TXNIP, were measured using quantitative real-time PCR and Western blot analyses. The effect of ethanol-induced TXNIP on the NLRP3 inflammasome was assessed in human macrophages transfected with TXNIP siRNA.ResultsU937 cells treated with 100 mM ethanol for 24 h induced NLRP3 and interleukin (IL)-1β expression. Ethanol increased reactive oxygen species generation in a time- and dose-dependent manner. AhR mRNA expression was downregulated in U937 cells treated with 100 mM ethanol, whereas CYP1A1 mRNA expression increased. Treatment with ethanol increased NLRP3 and IL-1β mRNA and protein expression in U937 cells exposed to 1.0 mg/mL of MSU crystals for 24 h. TXNIP expression in U937 cells incubated with both 100 mM ethanol and 1.0 mg/mL of MSU crystals was significantly higher than in cells incubated with MSU crystals alone. Treatment with 100mM ethanol for 24 h downregulated NLRP3 and IL-1β expression in MSU crystal-activated U937 cells transfected with TXNIP siRNA, compared to those with scramble siRNA.ConclusionEthanol stimulates uric acid-induced NLRP3 inflammasome activation through regression of AhR and upregulation of TXNIP.

Activation of aryl hydrocarbon receptor (AhR) in mesenchymal stem cells modulates macrophage polarization in asthma

Macrophage polarization has been demonstrated to exert a vital role on asthma pathogenesis. Mesenchymal stem cells (MSC) have the capacity to modulate macrophage differentiation from a pro-inflammatory M1 phenotype toward an anti-inflammatory M2 phenotype. However, the impact of MSC-macrophage interactions on asthma development and underlying mechanisms responsible for this interaction remain largely unknown. The aim of this study was to investigate the role of AhR expressed on MSC in macrophage polarization in a cockroach extract (CRE)-induced asthma mouse model. The studies here revealed that MSC polarized macrophages from a pro-inflammatory M1 phenotype toward an anti-inflammatory M2 phenotype in this model. The mRNA levels of interleukin (IL)-6, IL-1β, and NOS2 as M1 markers were significantly decreased while those of select M2 markers such as Arg-1, FIZZ1, and YM-1 were significantly enhanced. It was also observed that aryl hydrocarbon receptor (AhR) signaling was significantly increased during asthma pathogenesis as demonstrated by enhanced mRNA expression of AhR, CYP1a1, and CYP1b1. It was also seen that the elevated AhR signaling was able to attenuate the onset of asthma. Use of an AhR antagonist (CH223191) resulted in significant inhibition of the AhR signaling and increases in M2 marker expression, but led to elevation of expression of M1 markers in the CRE-induced asthma model. Taken together, the current study showed that MSC can modulate macrophage polarization, in part, via activation of AhR signaling during CRE-induced asthma.